Advanced Molecular Genetics by M. P. O’Connell, U. Priefer, N. Datta, M. E. Nugent (auth.),

By M. P. O’Connell, U. Priefer, N. Datta, M. E. Nugent (auth.), Alfred Pühler, Kenneth N. Timmis (eds.)

The improvement of robust new thoughts and refmements of tech­ niques in molecular genetics lately, and the surge in curiosity in biotechnology in keeping with genetic equipment, have heralded a brand new golden age in molecular genetics, and encouraged in various disciplines a lot curiosity within the applied sciences themselves and their power makes use of in simple and utilized biomedical sciences. even though a few very good professional laboratory manuals (especially the chilly Spring Harbor Laboratory manuals through I. H. Miller; R. W. Davies et al. ; and T. Maniatis et al. ) on definite chapters of molecular genetics exist, no basic textual content that covers a wide spectrum of the sub­ ject has to this point been released. the aim of this guide is to pre­ despatched such a lot, notwithstanding of necessity now not all the very important tools of molecular genetics, in a chain of straightforward experiments, lots of which are easily entire through the microbiologist, biochemist or biotechnologist that has had purely constrained publicity to genetics. the rest of the experiments require both higher familiarity with the topic, or advice by way of an individual with such adventure. The publication may still, hence, not just allow members to procure new proce­ dures for ongoing tasks, but in addition function a foundation for the educating of molecular genetic concepts in formal predoctoral and postdoctoral laboratory courses.

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D) Materials. . . . . . . . . . . . . . . . . . .. 7. References. . . . . . . . . . . . . . . . . . . . . . 38 39 39 39 39 40 40 40 40 41 44 45 45 46 46 47 47 47 48 48 49 50 1. General Introduction The experiments described below characterize antibiotic resistance plasmids that will replicate in E. coli K12. For other plasmids and plasmids of other bacterial species, methods must be adapted. No general instructions are possible for the characterization of any plasmid in any bacterial genus, although the availability of a plasmid-free I Dept.

1977) Jacob et aI. (1977) 3. Experiment 2: Conjugative Transfer of Plasmids to E. coli K12 a) Introduction A medium or technique must be devised to allow separation of K12 clones that have acquired a plasmid from a large population of wild-type donors and plasmid-free E. coli K12. This generally requires the recipient strain to differ from the wild-type donor by at least two characters, in addition to the putative plasmid character. One distinguishing character allows counter selection of the donor and another, unselected one distinguishes mutants of the donor from recipient clones that have received plasmid characters from the donor.

Minimal salts agar + glu, pro, met + drugs appropriate for selection of R transfer. 4. Experiment 3: Transfer of Plasmids to E. coli K12 by Transformation a) Introduction It is sometimes necessary or convenient to transfer resistance or other phenotypic markers by transformation. This is the case when transfer by direct conjugation or mobilization is not successful or to separate plasmids that have been transferred together by conjugation. The methods of transformation vary according to the bacterial genus; a review is given in this volume.

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